Blotting is a process used to transfer macromolecules from an electrophoresis matrix to a membrane for further analysis. Southern blotting is used for DNA analysis, Northern blotting is used for RNA analysis and Western blotting is used for protein analysis.
In Western blotting (or immunoblotting), proteins in a sample are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a membrane (e.g., nitrocellulose or polyvinylidene) by capillary action or electroblotting. After the proteins are immobilized on the surface of the membrane, the proteins are contacted with a primary antibody that specifically binds to a target protein. The bound primary antibody is then detected, for example, by contacting the primary antibody with a secondary antibody conjugated to a detectable label such as a fluorescent label. The detectable label is then visualized by optical techniques (i.e., techniques that measure emitted light).
Although immunoblotting provides useful information, the technique is time consuming, requires a high level of technical skill, is reagent intensive, has limited throughput, and is poorly suited for multiplexing.